Myosin II protein molecules, which consist of a pair of heavy chains (approximately 200 kDa) and two pairs of light chains (15-28 kDa), exist in all eukaryotic cells. Together with actin filaments, they produce contractile force mediated by ATP hydrolysis. While this contractile property is prominent in differentiated muscle tissues, it is also involved in diverse cellular motile processes such as cytokinesis, cell migration and cell adhesion in nonmuscle cells, as well as in undifferentiated muscle cells. In vertebrates, there are over 10 different myosin II isoforms, each of which contain different myosin II heavy chains (MHCs). MHC isoform diversity is generated by multiple genes as well as by alternative splicing of pre-mRNA. Previous studies have demonstrated cell type-specific expression of MHC isoforms as well as changes in MHC isoforms during the course of muscle and nervous tissue development. This research program has investigated the regulatory mechanisms responsible for the expression of two nonmuscle MHC genes, NMHC-A and NMHC-B. We have focused on NMHC-A gene regulation as it relates to cell type-dependent transcriptional mechanisms and NMHC-B gene regulation as it relates to neural cell-specific alternative pre-mRNA splicing mechanisms. With respect to NMHC-A gene regulation, we have previously identified three clustered cis-regulatory elements (A, C and F) in intron 1, which modulates transcription in a cell type-dependent manner. One of these elements (element F) contains an E-box sequence. In this study, we undertook to identify and characterize trans-acting factors responsible for element F-mediated regulation. Yeast one-hybrid screening allowed isolation of cDNAs encoding transcriptional factors TFEC, TFE3 and USF2, each of which contains basic helix-loop-helix and leucine zipper motifs. Furthermore, cDNA cloning by polymerase chain reaction yielded cDNAs for two TFEC isoforms, designated TFEC-l and -s, which are generated by alternative pre-mRNA splicing. In addition to these four factors, USF1, which is known to share the same DNA binding elements with USF2, was isolated for comparison. Electrophoretic mobility shift assays and cotransfection studies of the expression constructs with reporter gene constructs revealed that the above five factors have different binding activities for element F with different transactivation potencies. The results suggest that TFEC-l is a major factor, together with TFE3, for activation of the nonmuscle MHC-A transcription via element F. We also found that the N-terminal activation domain exists only in TFEC-1, whereas the C-terminal activation domain is common to both the l and s isoforms. This study provides the first evidence of TFEC being an activator of transcription, with two separate activation domains. With respect to NMHC-B, previous studies in this laboratory have demonstrated the existence of a neural cell-specific NMHC-B isoform, in addition to the ubiquitously expressed form of NMHC-B. This neural cell-specific isoform is generated by alternative splicing of a single cassette type of exon N30 which is located between constitutive exons E5 and E6. To characterize regulatory elements for alternative splicing of N30, neural retinoblastoma Y79 cells, which are capable of including N30 to a large extent during the postmitotic and differentiated states, were used as a model system. A cis-acting intronic enhancer (IDDE), located approximately 1.5 kb downstream of N30, and an exonic enhancer located within N30 were found to be required for neural cell-specific inclusion of N30. The IDDE activates the upstream E5-N30 splicing in vitro by facilitating early prespliceosome complex formation. In contrast, the IDDE has no effect on the downstream N30-E6 splicing where the IDDE resides. Inspection of splice site consensus sequences shows that a polypyrimidine (Py) tract preceding N30 is suboptimal for the binding of the essential splicing factor U2AF. Optimizing this Py tract completely relieves the requirement for the IDDE in in vitro and in situ (transfected minigene transcript) splicing of N30. Furthermore, overexpression of truncated U2AF65 selectively inhibits IDDE-mediated N30 inclusion in mRNA from the wild-type minigene in a dominant negative fashion. These results support the hypothesis that the IDDE promotes usage of the 3 splice sites preceding N30 by a network of protein-protein interactions implicated in the recruitment of U2AF to a suboptimal Py tract.